This invention relates to pharmaceutically active compounds which inhibit FabI and are useful for the treatment of bacterial infections.
While the overall pathway of saturated fatty acid biosynthesis is similar in all organisms, the fatty acid synthase (FAS) systems vary considerably with respect to their structural organization. Vertebrates and yeast possess a FAS in which all the enzymatic activities are encoded on one or two polypeptide chains, respectively, and the acyl carrier protein (ACP) is an integral part of the complex. In contrast, in bacterial FAS, each of the reactions is catalyzed by a distinct, mono-functional enzyme and the ACP is a discrete protein. Therefore, there is considerable potential for the selective inhibition of the bacterial system by antibacterial agents.
FabI (previously designated EnvM) functions as an enoyl-ACP reductase (Bergler, et al, (1994), J. Biol. Chem. 269, 5493-5496) in the final step of the four reactions involved in each cycle of bacterial fatty acid biosynthesis. In this pathway, the first step is catalyzed by xcex2-ketoacyl-ACP synthase, which condenses malonyl-ACP with acetyl-CoA (FabH, synthase III). In subsequent rounds, malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II, respectively). The second step in the elongation cycle is ketoester reduction by NADPH-dependent xcex2-ketoacyl-ACP reductase (FabG). Subsequent dehydration by xcex2-hydroxyacyl-ACP dehydrase (either FabA or FabZ) leads to trans-2-enoyl-ACP, which in turn is converted to acyl-ACP by NADH-dependent enoyl-ACP reductase (FabI). Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP (16C), where upon the cycle is stopped largely due to feedback inhibition of FabI by palmitoyl-ACP (Heath, et al, (1996), J. Biol. Chem. 271, 1833-1836). Thus, FabI is a major biosynthetic enzyme and is a key regulatory point in the overall synthetic pathway of bacterial fatty acid biosynthesis. Therefore, FabI is an ideal target for antibacterial intervention.
Studies have shown that diazaborine antibiotics inhibit fatty acid, phospholipid and lipopolysaccharide (LPS) biosynthesis and that the antibacterial target of these compounds is FabI. For example, derivative 2b18 from Grassberger, et al (1984) J. Med Chem 27 947-953 has been reported to be a non-competitive inhibitor of FabI (Bergler, et al, (1994), J. Biol. Chem. 269, 5493-5496). Also, plasmids containing the FabI gene from diazaborine resistant S. typhimurium conferred diazaborine resistance in E. coli (Turnowsky, et al, (1989), J. Bacteriol., 171, 6555-6565). Furthermore, inhibition of FabI either by diazaborine or by raising the temperature in a FabI temperature sensitive mutant is lethal. These results demonstrate that FabI is essential to the survival of the organism (Bergler, et al, (1994), J. Biol. Chem. 269, 5493-5496).
Recent studies have shown that FabI is also the target for the broad spectrum antibacterial agent triclosan (McMurry, et al, (1998) Nature 394, 531-532). A crystal structure of the E. Coli FabI complexed with NAD and triclosan shows that triclosan acts as a site-directed, very potent inhibitor of FabI by mimicking its natural substrate (Levy, et al, (1999) Nature 398, 383-384). Ward. et al ((1999) Biochem. 38, 12514-12525) have shown that there is no evidence for the formation of a covalent complex between FabI and triclosan, which would be analogous to the diazaborines: triclosan differs from these compounds in that it is a reversible inhibitor of FabI. The structural data for the complex of FabI with NAD and triclosan provides important information about FabI as a therapeutic target.
Importantly, it has now been discovered that certain compounds are FabI inhibitors and have antibacterial activity, and, therefore, may be useful for the treatment of bacterial infections in mammals, particularly in man.
This invention comprises compounds of the formula (I), as described hereinafter, which inhibit FabI and are useful in the treatment of bacterial infections.
This invention is also a pharmaceutical composition comprising a compound according to formula (I) and a pharmaceutically acceptable carrier.
This invention is also a method of treating bacterial infections by inhibiting FabI. In a particular aspect, the compounds of this invention are useful as antibacterial agents.
This invention comprises compounds of formula (I) and (II): 
wherein:
R1 is H or C1-4alkyl;
R2 is C1-4alkyl;
R3 is 
Q is a single bond, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94CH2xe2x80x94, or xe2x80x94CH2CH2xe2x80x94;
R4 is C1-6alkyl, OC1-6alkyl, OH, CF3 or piperidinyl; 
xe2x80x83is a five- or six-membered heteroaromatic ring or a six-membered aromatic ring unsubstituted or substituted by R5; 
xe2x80x83is a five- or six-membered heteroaromatic ring or a six-membered aromatic ring unsubstituted or substituted by R6; 
xe2x80x83is a five- or six-membered heteroaromatic ring or a six-membered aromatic ring unsubstituted or substituted by R6;
R5 is ORxe2x80x2 or NRxe2x80x2C(O)Rxe2x80x2;
R6 is ORxe2x80x2 or N(Rxe2x80x2)2;
X is H, C1-4alkyl, ORxe2x80x2, SRxe2x80x2, CN, N(Rxe2x80x2)2, CH2N(Rxe2x80x2)2, NO2, CF3, CO2Rxe2x80x2, CON(Rxe2x80x2)2, CORxe2x80x2, NRxe2x80x2C(O)Rxe2x80x2, F, Cl, Br, I or xe2x80x94S(O)rCF3;
Rxe2x80x2 is H, C1-6alkyl or xe2x80x94C0-6alkyl-Ar; and
r is 0, 1 or 2;
or a pharmaceutically acceptable salt thereof.
Also included in this invention are pharmaceutically acceptable addition salts and complexes of the compounds of this invention. In cases wherein the compounds of this invention may have one or more chiral centers, unless specified, this invention includes each unique racemic compound, as well as each unique nonracemic compound.
In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, such as 
and 
each tautomeric form is contemplated as being included within this invention, whether existing in equilibrium or locked in one form by appropriate substitution with Rxe2x80x2. The meaning of any substituent at any one occurrence is independent of its meaning, or any other substituent""s meaning, at any other occurrence.
Also included in this invention are prodrugs of the compounds of this invention. Prodrugs are considered to be any covalently bonded carriers which release the active parent drug according to formula (I) in vivo.
The compounds of formula (I) inhibit FabI. Inhibition of this enzyme is useful in the treatment of bacterial infections. Also, the compounds of this invention may be useful as antifungal agents. Additionally, the compounds may be useful in combination with known antibiotics.
With respect to formula (I) and (II), this invention preferably includes compounds of formula (Ia) and (IIa): 
in which R3 is as defined for formula (I) and (II) compounds.
Suitably, with respect to formula (I) and (II), R3 is: 
Preferably, 
is 2-, 3- or 4-pyridyl, 2- or 3-furanyl, 2- or 3-thiophenyl, thiazolyl, phenyl or phenyl substituted by OC1-4alkyl or NHC(O)C1-4alkyl. Most preferably, 
is 3- or 4-pyridyl, 3-furanyl, 3-thiophenyl, phenyl or phenyl substituted by OCH3 or NHC(O)CH3. Also, preferably, 
is 2-, 3- or 4-pyridyl, phenyl or phenyl substituted by OH or NH2. Most prefarably, 
is phenyl or 2-pyridyl.
Suitably, with respect to formula (I) and (II), R3 is: 
in which Q is a single bond, xe2x80x94Oxe2x80x94 or CH2CH2xe2x80x94. Preferably, Q is a single bond.
Suitably, with respect to formula (I), R3 is: 
Preferably, 
is phenyl unsubstituted or substituted by OH, NH2 or N(CH3)2 and R4 is C1-4alkyl, OC1-4alkyl, OH, CF3 or piperidinyl.
Representative of the novel compounds of this invention are the compounds of the examples hereinafter.
Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of this invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochem., 158, 9 (1984).
C1-4alkyl as applied herein means an optionally substituted alkyl group of 1 to 4 carbon atoms, and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl. C1-6alkyl additionally includes pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. C0-4alkyl and C0-6alkyl additionally indicates that no alkyl group need be present (e.g., that a covalent bond is present).
Any C1-4alkyl or C1-6alkyl may be optionally substituted with the group Rx, which may be on any carbon atom that results in a stable structure and is available by conventional synthetic techniques. Suitable groups for Rx are C1-4alkyl, ORxe2x80x2, SRxe2x80x2, CN, N(Rxe2x80x2)2, CH2N(Rxe2x80x2)2, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94CO2Rxe2x80x2xe2x80x94CON(Rxe2x80x2)2, xe2x80x94CORxe2x80x2xe2x80x94NRxe2x80x2C(O)Rxe2x80x2, F, Cl, Br, I, or xe2x80x94S(O)rCF3, wherein Rxe2x80x2 and r are as defined for formula (I) compounds.
Halogen or halo means F, Cl, Br, and I.
Ar, or aryl, as applied herein, means phenyl or naphthyl, or phenyl or naphthyl substituted by one to three substituents, such as those defined above for alkyl, or substituted by methylenedioxy.
Het, or heterocycle, indicates an optionally substituted five or six membered monocyclic ring, or a nine or ten-membered bicyclic ring containing one to three heteroatoms chosen from the group of nitrogen, oxygen and sulfur, which are stable and available by conventional chemical synthesis. Illustrative heterocycles are benzofuryl, benzimidazolyl, benzopyranyl, benzothienyl, furyl, imidazolyl, indolinyl, morpholinyl, piperidinyl, piperazinyl, pyrrolyl, pyrrolidinyl, tetrahydropyridinyl, pyridinyl, thiazolyl, thienyl, quinolinyl, isoquinolinyl, and tetra- and perhydro-quinolinyl and isoquinolinyl. Any accessible combination of up to three substituents on the Het ring, such as those defined above for alkyl, that are available by chemical synthesis and are stable are within the scope of this invention.
Certain radical groups are abbreviated herein. t-Bu refers to the tertiary butyl radical, Boc refers to the t-butyloxycarbonyl radical, Fmoc refers to the fluorenylmethoxycarbonyl radical, Ph refers to the phenyl radical, Cbz refers to the benzyloxycarbonyl radical, Bn refers to the benzyl radical, Me refers to methyl, Et refers to ethyl, Ac refers to acetyl, Alk refers to C1-4alkyl, Nph refers to 1- or 2-naphthyl and cHex refers to cyclohexyl. Tet refers to 5-tetrazolyl.
Certain reagents are abbreviated herein. DCC refers to dicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, EDC refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride, HOBt refers to 1-hydroxybenzotriazole, THF refers to tetrahydrofuran, DIEA refers to diisopropylethylamine, DEAD refers to diethyl azodicarboxylate, PPh3 refers to triphenylphosphine, DIAD refers to diisopropyl azodicarboxylate, DME refers to dimethoxyethane, DMF refers to dimethylformamide, NBS refers to N-bromosuccinimide, Pd/C refers to a palladium on carbon catalyst, PPA refers to polyphosphoric acid, DPPA refers to diphenylphosphoryl azide, BOP refers to benzotriazol-1-yloxy-tris(dimethyl-amino)phosphonium hexafluorophosphate, HF refers to hydrofluoric acid, TEA refers to triethylamine, TFA refers to trifluoroacetic acid, PCC refers to pyridinium chlorochromate.
Generally, the compounds of formula (I) and (II) are prepared by:
(i) reacting a compound of formula (III) with a compound of formula (IV): 
wherein R1, R2, R3 and X are as defined in formula (I), with any reactive functional groups protected, in the presence of EDC and HOBT; or
(ii) reacting a compound of formula (V) with a compound of formula (IV): 
wherein R1, R2, R3 and X are as defined in formula (I), with any reactive functional groups protected, in the presence of EDC and HOBT;
and thereafter removing any protecting groups, and optionally forming a pharmaceutically acceptable salt.
In particular, compounds of the formula (I) and (II) are prepared by the general methods described in Schemes hereinafter. 
(a) 1-methyl-2-(methylaminomethyl)indole, EDC, HOBt.H2O, Et3N, DMF; (b) furan-3-boronic acid. Pd(PPh3)4, Cs2CO3, DME, H2O.
A suitable 2-haloarylcarboxylic acid, for instance 2-bromobenzoic acid (I-1), is converted to an activated form using, for example, EDC and HOBt, or SOCl2, and the activated form is subsequently reacted with an appropriate amine, for instance 1-methyl-2-(methylaminomethyl)indole, in a suitable solvent such as DMF, CH2Cl2, or CH3CN, to afford I-2. Depending on whether acid neutralization is required, an added base, such as triethylamine (Et3N), diisopropylethylamine ((i-Pr)2NEt), or pyridine, may be used. Many additional methods for converting a carboxylic acid to an amide are known, and can be found in standard reference books, such as xe2x80x9cCompendium of Organic Synthetic Methodsxe2x80x9d, Vol. I-VI (published by Wiley-Interscience), or Bodansky, xe2x80x9cThe Practice of Peptide Synthesisxe2x80x9d (published by Springer-Verlag). The resulting haloarylcarboxamide 1-2 is reacted with an appropriate boronic acid derivative, such as furan-3-boronic acid, to afford I-3 in a Suzuki-type reaction (Synth. Commun. 1981, 11, 513; for a review, see Martin, A. R.; Yang, Y. Acta Chem. Scand. 1993, 47, 221). This reaction is generally mediated by tetrakis(triphenylphosphine)palladium(0), although a wide variety of palladium catalysts can be used, including palladium acetate, palladium acetate in the presence of tri(ortho-tolyl)phosphine, bis(triphenylphosphine)palladium(II) chloride, 1,4-bis(diphenylphosphinobutane)palladium(II) chloride, bis(acetonitrile)palladium(II) chloride, bis(dibenzylideneacetone)palladium(0), and palladium on carbon in the presence of triphenylphosphine. The reaction requires a base as an acid scavenger, and generally aqueous sodium carbonate (Na2CO3) is used. However, other bases, such as sodium hydrogen carbonate, potassium carbonate, cesium carbonate, cesium fluoride, and triethylamine can be used. Typically, water-miscible neutral solvents, including DME, THF, and DMF, are preferred. 
(a) para-anisaldehyde, NaH, tert-BuOH, DMF; (b) H2, Pd/C, NaOH, H2O; (c) 1-methyl-2-(methylaminomethyl)indole, EDC, HOBt.H2O, (i-Pr)2NEt, DMF.
According to the general procedure of Brenner et al. (J. Het. Chem. 1982, 19, 897-900), a suitable 2-methylnicotinic ester, for instance ethyl 2-methylnicotinate (II-1), is reacted with an aromatic aldehyde, such as para-anisaldehyde, to afford I-2. The reaction is mediated by strong base, typically sodium tert-butoxide, and is conducted in a polar solvent, generally DMF. Reduction of the olefin group of I-2 is conveniently accomplished by hydrogenation as described by van der Stelt, et al. (Arzneim.-Forsch. 1968, 18, 756-758). The reaction is conducted under basic conditions in the presence of a palladium catalyst, generally palladium on activated charcoal. Alkali metal hydroxides, such as sodium hydroxide or potassium hydroxide, are the preferred bases, and the reaction is typically conducted in H2O, although an aqueous hydroxylic solvent, such as aqueous MeOH, EtOH, or i-PrOH, may also be used. The resulting acid, II-3, is converted to II-4 by the methods described in Scheme I. 
(a) phenol, NaOMe, MeOH, then Cu, DMA; (b) H2, Pd/C, MeOH; (c) 1-methyl-2-(methylaminomethyl)indole, EDC, HOBt.H2O, Et3N, DMF.
A suitable 2-halobenzoic acid derivative, for instance 2-chloro-4-nitrobenzoic acid (III-2), reacts with an appropriate alcohol, for example phenol, to afford an ether derivative such as III-2. The reaction is mediated by copper powder, and is typically conducted in a high-boiling, polar, aprotic solvent, such as N,N-dimethylacetamide (DMA). The alcohol derivative is oftentimes converted to the corresponding alkoxide by deprotonation with a suitable base. Typical bases include sodium hydride (NaH) in THF, DMF, or DMA (or mixtures thereof), or sodium methoxide (NaOMe) in methanol. The nitro group of III-2 can be reduced conveniently by hydrogenation over a palladium metal catalyst, typically palladium on activated carbon (Pd/C). Suitable solvents for this reaction include MeOH, EtOH, EtOAc, AcOH, or mixtures thereof. Other methods for the reduction of nitro groups to the corresponding amine are known to those of skill in the art, and can be found in standard reference volumes, such as Compendium of Organic Synthetic Methods (Wiley-Interscience). The resulting derivative III-3 is converted to III-4 as described in Scheme I. 
(a) 3-(acetylamino)phenol, Cu2O, DMA; (b) NaOH, dioxane, H2O, then HCl; (c) 1-methyl-2-(methylaminomethyl)indole, EDC, HOBt.H2O, Et3N, DMF.
Alternatively, a suitable 2-halobenzoic acid derivative, for instance methyl 2-iodobenzoate (IV-1), reacts with an appropriate alcohol, for example 3-(acetylamino)phenol, in the presence of a copper(I) salt, preferably copper(I) oxide (Cu2O), to afford the ether derivative IV-2. A polar, aprotic solvent such as N,N-dimethylacetamide (DMA) is preferred. The methyl ester of IV-2 is hydrolyzed using aqueous base, for example, LiOH or NaOH in aqueous dioxane, THF, methanol or ethanol, and the intermediate carboxylate salt is acidified with a suitable acid, for instance TFA or HCl, to afford the carboxylic acid IV-3, which is converted to IV-4 as described in Scheme I.
Amide coupling reagents as used herein denote reagents which may be used to form peptide bonds. Typical coupling methods employ carbodiimides, activated anhydrides and esters and acyl halides. Reagents such as EDC, DCC, DPPA, PPA, BOP reagent, HOBt, N-hydroxysuccinimide and oxalyl chloride are typical.
Acid addition salts of the compounds are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric, acetic, tnifluoroacetic, maleic, succinic or methanesulfonic. Certain of the compounds form inner salts or zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine. Cations such as Li+, Na+, K+, Ca++, Mg++ and NH4+ are specific examples of cations present in pharmaceutically acceptable salts.
This invention also provides a pharmaceutical composition which comprises a compound according to formula (I) or (II) and a pharmaceutically acceptable carrier. Accordingly, the compounds of formula (I) or (II) may be used in the manufacture of a medicament. Pharmaceutical compositions of the compounds of formula (I) or (II) prepared as hereinbefore described may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation may be a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
Alternately, these compounds may be encapsulated, tableted or prepared in a emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
For rectal administration, the compounds of this invention may also be combined with excipients, such as cocoa butter, glycerin, gelatin or polyethylene glycols, and molded into a suppository.
For topical administration, the compounds of this invention may be combined with diluents to take the form of ointments, gels, pastes, creams, powders or sprays. The compositions which are ointments, gels, pastes or creams contain diluents, for example, animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures of these substances. The compositions which are powders or sprays contain diluents, for example, lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Additionally, for topical ophthalmologic administration, the typical carriers are water, mixtures of water and water miscible solvents, such as lower alkanols or vegetable oils, and water-soluble non-toxic polymers, for example cellulose derivatives, such as methyl cellulose.
The compounds described herein are inhibitors of FabI, and are useful for treating bacterial infections. For instance, these compounds are useful for the treatment of bacterial infections, such as, for example, infections of upper respiratory tract (e.g. otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g. empyema, lung abscess), cardiac (e.g. infective endocarditis), gastrointestinal (e.g. secretory diarrhoea, splenic abscess, retroperitoneal abscess), CNS (e.g. cerebral abscess), eye (e.g. blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g. epididymitis, intrarenal and perinephric abscess, toxic shock syndrome), skin (e.g. impetigo, folliculitis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis), and bone and joint (e.g. septic arthritis, osteomyelitis). Also, the compounds of this invention may be useful as antifungal agents. Additionally, the compounds may be useful in combination with known antibiotics.
The compounds of this invention are administered to the patient, in a manner such that the concentration of drug is sufficient to treat bacterial infections. The pharmaceutical composition containing the compound is administered at an oral dose of between about 10 mg to about 1000 mg, taken once or several times daily, in a manner consistent with the condition of the patient. Preferably, the oral dose would be about 50 mg to about 500 mg, although the dose may be varied depending upon the age, body weight and symptoms of the patient. For acute therapy, parenteral administration is preferred. An intravenous infusion of the compound of formula (I) or (II) in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. The precise level and method by which the compounds are administered is readily determined by one skilled in the art.
The compounds may be tested in one of several biological assays to determine the concentration of compound which is required to have a given pharmacological effect.
Cloning of S. aureus FabI:
The fabI gene was cloned from the chromosomal DNA of S. aureus strain WCUH29 using the polymerase chain reaction. Amplification was performed using Taq DNA polymerase (BRL) and the following primers: 5xe2x80x2-CGCCTCGAGATGTFAAATCTGAAAACAAAACATATGTC-3xe2x80x2 and 5xe2x80x2-CGCGGATCCAATCAAGTCAGGTGAAATATCCA-3xe2x80x2 (XhoI and BamHI sites underlined). The resulting fragment was then digested with XhoI and BamHII and ligated into XhoI- and BamHI-digested expression vector pET-16b (Novagen), producing pET-His10-fabI. The gene sequence of fabI was confirmed by automated cycle sequencing using an Applied Biosystems model 377 machine. The untagged version of pET-fabI was constructed by digesting pET-His10-fabI with NcoI and NdeI to remove a 97 bp fragment encoding the His 10 tag, the factor Xa cleavage site and the first 8 amino acids of FabI, and replacing it with a linker encoding the first 8 amino acids of FabI plus a glycine residue between the initiator methionine and the lysine at position 2. This plasmid was called pET-fabI. The linker was made by annealing the following two oligonucleotides: 5xe2x80x2-CATGGGCTTAAATCTTGAAAACAAAACA-3xe2x80x2 and 5xe2x80x2-TATGTTTTGTTTTCAAGATTTAAGCC-3xe2x80x2. The linker sequence in pET-fabI was confirmed by dideoxy sequencing. Only native FabI was used for compound evaluation. For overproduction of native FabI, plasmid pET-fabI was transformed into BL21(DE3) (Novagen) cells, to form strain BL21(DE3):pET-fabI.
Purification of S. aureus FabI
S. aureus FabI was expressed as soluble protein to 10% of total cell protein, 400 g cells being recovered from 15 L fermentation in tryptone phosphate medium. The cells were lysed and the sample centrifuged. The resulting supernatant was filtered and purified using three consecutive chromatography columns: ion-exchange (Sourse 15Q), dye-affinity (Blue sepharose), and size exclusion chromatography columns (Superose 12). After each column the FabI containing fractions were pooled, concentrated, and checked for purity and biological activity.
Cloning of E. coli FabI:
A PCR fragment of correct size for E. coli FabI was PCR amplified from E. coli chromosomal DNA, subcloned into the TOPO TA cloning vector, and verified by colony PCR+restriction endonuclease analysis. The presumptive E. coli FabI PCR fragment was subcloned into the expression vector pBluePet. The FabI clone was transformed into E. coli strain BL21(DE3). Small Scale expression studies show an over-expressed protein band of correct molecular weight (xcx9c28 Kda) for E. coli FabI clearly visible following Coomassie staining of SDS PAGE gels. DNA sequencing of the E. coli FabI expression constructs illustrated that no errors were apparent. Nxe2x80x2 terminal amino acid sequencing has confirmed the over-expressed protein band to be E. coli FabI.
Purification of E. coli FabI
E. coli FabI was expressed as soluble protein to 15% of total cell protein, 120 g cells being recovered from 3 L fermentation in shake flasks in modified terrific broth. The cells were lysed and the sample centrifuged. The resulting supernatant was filtered and purified using three consecutive chromatography columns: ion-exchange (Sourse 15Q), dye-affinity (blue sepharose), and size exclusion (superose 12). After each column the FabI containing fractions were pooled, concentrated and checked for purity and biological activity.
S. aureus FabI Enzyme Inhibition Assay (NADH):
Assays were carried out in half-area, 96-well microtitre plates. Compounds were evaluated in 50-uL assay mixtures containing 100 mM NaADA, pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol, 0.25 mM crotonoyl CoA, 1 mM NADH, and an appropriate dilution of S. aureus FabI. Inhibitors were typically varied over the range of 0.01-10 uM. The consumption of NADH was monitored for 20 minutes at 30xc2x0 C. by following the change in absorbance at 340 nm. Initial velocities were estimated from an exponential fit of the non-linear progress curves represented by the slope of the tangent at t=0 min. IC50""s were estimated from a fit of the initial velocities to a standard, 4-parameter model and are typically reported as the meanxc2x1S.D. of duplicate determinations. Triclosan, a commercial antibacterial agent and inhibitor of FabI, is currently included in all assays as a positive control. Compounds of this invention have IC50""s from about 1.50 micromolar to about 0.15 micromolar.
S. aureus FabI Enzyme Inhibition Assay (NADPH):
Assays were carried out in half-area, 96-well microtitre plates. Compounds were evaluated in 150-uL assay mixtures containing 100 mM NaADA, pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol, 0.25 mM crotonoyl CoA, 50 uM NADPH, and an appropriate dilution of S. aureus FabI. Inhibitors were typically varied over the range of 0.01-10 uM. The consumption of NADPH was monitored for 20 minutes at 30xc2x0 C. by following the change in absorbance at 340 nm. Initial velocities were estimated from an exponential fit of the non-linear progress curves represented by the slope of the tangent at t=0 min. IC50""s were estimated from a fit of the initial velocities to a standard, 4-parameter model and are typically reported as the meanxc2x1S.D. of duplicate determinations. Triclosan, a commercial antibacterial agent and inhibitor of FabI, is currently included in all assays as a positive control.
E. coli FabI Enzyme Inhibition Assay:
Assays were carried out in half-area, 96-well microtitre plates. Compounds were evaluated in 150-uL assay mixtures containing 100 mM NaADA, pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol, 0.25 mM crotonoyl CoA, 50 uM NADH, and an appropriate dilution of E. coli FabI. Inhibitors were typically varied over the range of 0.01-10 uM. The consumption of NADH was monitored for 20 minutes at 30xc2x0 C. by following the change in absorbance at 340 nm. Initial velocities were estimated from an exponential fit of the non-linear progress curves represented by the slope of the tangent at t=0 min. IC50""s were estimated from a fit of the initial velocities to a standard, 4-parameter model and are typically reported as the meanxc2x1S.D. of duplicate determinations. Triclosan, a commercial antibacterial agent and inhibitor of FabI, is currently included in all assays as a positive control. Compounds of this invention have IC50""s from about 20.0 micromolar to about 2.0 micromolar.
Preparation and Purification of Crotonoyl-ACP:
Reactions contained 5 mg/mL E. coli apo-ACP, 0.8 mM crotonoyl-CoA (Fluka), 10 mM MgCl2, and 30 uM S. pneumoniae ACP synthase in 50 mM NaHEPES, pH 7.5. The mixture was gently mixed on a magnetic stirrer at 23xc2x0 C. for 2 hr, and the reaction was terminated by the addition of 15 mM EDTA . The reaction mixture was filtered through a 0.2 micron filter (Millipore) and applied to a MonoQ column (Pharmacia) equilibrated with 20 mM Tris-Cl, pH 7.5. The column was washed with buffer until all non-adherent material was removed (as observed by UV detection), and the crotonoyl-ACP was eluted with a linear gradient of 0 to 400 mM NaCl.
S. aureus FabI Enzyme Inhibition Assay Using Crotonoyl-ACP:
Assays are carried out in half-area, 96-well microtitre plates. Compounds are evaluated in 150 uL assay mixtures containing 100 mM NaADA, pH 6.5 (ADA=N-(2-acetamido)-2-iminodiacetic acid), 4% glycerol, 25 uM crotonoyl-ACP, 50 uM NADPH and an appropriate dilution of S. aureus FabI (approximately 20 nM). Inhibitors are typically varied over the range of 0.01-10 uM. The consumption of NADPH is monitored for 20 minutes at 30xc2x0 C. by following the change in absorbance at 340 nm. Initial velocities are estimated from a linear fit of the progress curves. IC50""s are estimated from a fit of the initial velocities to a standard, 4-parameter model (Equation 1) and are typically reported as the meanxc2x1S.D. of duplicate determinations. The apparent Ki is calculated from Equation 2 assuming the inhibition is competitve with crotonoyl-ACP.
v=Range/(1+[I]/IC50)s+Backgroundxe2x80x83xe2x80x83Equation 1:
Ki(app)=IC50/(1+[S]/Ks)xe2x80x83xe2x80x83Equation 2:
Antimicrobial Activity Assay:
Whole-cell antimicrobial activity was determined by broth microdilution using the National Committee for Clinical Laboratory Standards (NCCLS) recommended procedure, Document M7-A4, xe2x80x9cMethods for Dilution Susceptibility Tests for Bacteria that Grow Aerobicallyxe2x80x9d. The compound was tested in serial two-fold dilutions ranging from 0.06 to 64 mcg/mL. Test organisms were selected from the following laboratory strains: Staphylococcus aureus Oxford, Staphylococcus aureus WCUH29, Streptococcus pneumoniae ERY2, Streptococcus pneumoniae 1629, Streptococcus pneumoniae N 1387, Enterococcus faecalis I, Enterococcus faecalis 7, Haemophilus influenzae Q1, Haemophilus influenzae NEMC1, Moraxella Catarrhalis 1502, Escherichia coli 7623 AcrABEFD+, Escherichia coli 120 AcrABxe2x88x92, Escherichia coli MGJ655, Escherichia coli MG1658. The minimum inhibitory concentration (MIC) was determined as the lowest concentration of compound that inhibited visible growth. A mirror reader was used to assist in determining the MIC endpoint.
One skilled in the art would consider any compound with a MIC of less than 256 xcexcg/mL to be a potential lead compound. Preferably, the compounds used in the antimicrobial assays of the present invention have a MIC value of less than 128 xcexcg/mL. Most preferably, said compounds have a MIC value of less than 64 xcexcg/mL.